Download Neuropeptide Y immunoreactive neurons in the guinea

October 1984
Vol. 25/10
Investigative Ophthalmology
& Visual Science
A Journal of Dosic and Clinical Research
Articles
Neuropeptide Y Immunoreactive Neurons in the
Guinea-Pig Uveo and Retina
A. Druun, D. Ehinger, F. Sunder, K. Tornqvisr, and R. Uddmon
Neuropeptide Y (NPY) is a recently discovered, amidated 36 amino acid residue neuropeptide present
in many but not all sympathetic noradrenergic neurons. In the guinea-pig eye, NPY immunoreactive
fibers were found to have the same distribution as noradrenergic fibers except that there were fewer
at the iris dilator, in the cornea, and in the chamber angle. In the anterior uvea, the NPY
immunoreactive fibers disappeared after excision of the homolateral superior cervical sympathetic
ganglion, whereas in the choroid, many NPY immunoreactive fibers remained, indicating that they
originate elsewhere. NPY immunoreactivity thus is not found in all sympathetic adrenergic neurons
nor is it found only in such nerve fibers. In the retina, NPY immunoreactive fibers formed a single
layer of processes in sublamina 1 of the inner plexiform layer. NPY immunoreactive cell bodies were
found in the innermost cell row of the inner nuclear layer. The immunoreactivity was concentrated to
the hillock region of these cells. Invest Ophthalmol Vis Sci 25:1113-1123, 1984
NPY has been shown to occur in certain sympathetic, noradrenergic neurons.6'8 The internal eye
muscles are classical organs for studies on the autonomic nervous system, but the distribution of NPY
in them is unknown. We therefore have examined
the distribution and origin of nerve fibers with NPY
immunoreactivity in the uvea of the guinea-pig eye.
Further, the retina is an extensively analyzed and
easily accessible piece of CNS tissue that contains
several other neuropeptides, 10 " and we therefore
have included it in the study.
A number of peptides have in recent years attracted
attention as putative neurotransmitters or neuromodulators. Many of them were isolated originally from
the pancreas or the gastrointestinal tract and were
shown to occur also in the brain. Several of them
turned out to be alpha-amidated at the C-terminus,
and this prompted Tatemoto and Mutt1 to search for
additional such peptides in extracts from pig gut and
brain. A peptide with 36 amino acid residues, named
neuropeptide Y (NPY), was found to be present in
significant amounts in the brain of several animal
species.23 It also was found in many peripheral tissues
where it had distinct, biologic effects.4"8 NPY shows
considerable structural resemblance with pancreatic
polypeptide and peptide YY, and it was proposed
that these peptides form a special family of regulatory
peptides.2 The amino acid sequences of these peptides
have, been published.9
Materials and Methods
The eyes of 10 adult (300-400 g) outbred guineapigs of both sexes were used. Most animals were
pigmented, but a few albinotic ones also were examined because the dark melanophores in the pigmented
eyes could be suspected to disguise some nerve fibers.
However, no differences were discernible between
pigmented and albinotic eyes.
Sympathetic denervation was achieved in three
animals by excision of the cervical sympathetic ganglion chain on one side, all the way up to the base
of the skull. The other side served as control.
The animals were killed by an overdose of diethyl
ether, and the eyes were dissected out rapidly, bisected
and fixed for 24 hr at +4°C in 4% formaldehyde
buffered to pH 7.2 in a 0.1 M phosphate buffer. The
From the Departments of Ophthalmology and Histology, University of Lund, Lund, Sweden, and the Department of Oto-RhinoLaryngology at Malmo, Malmo Allmanna Sjukhus, Malmo, Sweden.
Supported by grants from the Helfrid and Lorentz Nilssons
Stiftelse, the H. Jarnhardts Stiftelse, the Thorsten and Elsa, Segerfalks
Stiftelse, the Carmen and Bertil Regners Stiftelse, and the Swedish
Medical Research Council (projects 14X-2321 and 14X-4499).
Submitted for publication: December 20, 1983.
Reprint requests: Ms. Anitha Bruun, Department of Ophthalmology, Research Department B, Lasarettet i Lund, S-22185 Lund,
Sweden.
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Fig. 1. Adrenergic fibers in the
iris (I) and ciliary processes (CP)
from a normal, albino, guinea pig,
demonstrated with the formaldehyde fluorescence method of Falck
and Hillarp. Adrenergic fibers occur
in front of the dilator (D), in the
iris stroma and in the stroma of the
ciliary processes. The picture is included in order to facilitate comparison with the NPY immunoreactive fibers. (Coronary section,
XI40).
tissue pieces were washed at +4°C for 2 days in
several changes of the phosphate buffer containing
30% sucrose. Fifteen micrometer sections were obtained in a cryostat, melted on the microscope slides,
and air-dried.
The NPY immunoreactivity was demonstrated with
the indirect immunofluorescence method of Coons
et al.12 The NPY antiserum (code no. NPYY/2, used
in dilution 1:400) was a kind gift from Dr. Piers
Emson, MRC Neuropharmacology Unit, Cambridge,
England. It has been shown to cross-react with other
peptides of the PP family in gut endocrine cells,
which seems due to minority antibody populations.8
Specificity controls were in the present work obtained
by preincubating diluted antiserum at +4°C overnight
with NPY, avian pancreatic polypeptide (APP), bovine
pancreatic polypeptide (BPP) or peptide YY (PYY),
all 100 Mg/ml diluted antiserum. No staining was
seen with antiserum that had been inactivated with
NPY, whereas absorption with APP, BPP, and PYY
did not influence appreciably the demonstrability of
the NPY immunoreactivity in the tissue. APP, BPP,
and PYY thus are not likely to be responsible for the
NPY-like immunofluorescence seen in this work.
Nevertheless, cross-reactions with unidentified peptides displaying the same antigenic site as NPY to
the antibody cannot be excluded. Therefore, the
structures revealed with the NPY antibody are referred
to in this paper as NPY immunoreactive fibers or .
NPY immunoreactive neurons.
For comparison, the adrenergic fibers of the guineapig eye were demonstrated with the formaldehyde
histofluorescence method of Falck and Hillarp.13
The animals used in this study have all been treated
according to the ARVO Resolution on the Use of
Animals in Research.
Results
Numerous NPY immunoreactive fibers were seen
in all parts of the uvea. They were thin and had
characteristic little oval beads (1-3 ^m long, 1-2 nm
across) at fairly regular intervals (about every 8-15
tim). The fluorescence intensity was often somewhat
less in the intervaricose part than in the varicosity.
In general, the uveal fibers seemed somewhat less
immunoreactive than fibers around extraocular ciliary
arteries or vessels occasionally encountered in extraocular muscles.
The appearance of the NPY immunoreactive fibers
corresponds precisely to that seen in adrenergic peripheral neurons as demonstrated with the formaldehyde fluorescence method.14"17 In order to facilitate
comparisons of the distribution of the adrenergic and
NPY immunoreactive fibers, Figures 1-3 show the
distribution of the noradrenergic fibers in the guineapig iris, ciliary processes, chamber angle, and retina.
More detailed descriptions have been published.14
The results of the current reexamination corresponded
well with the previous results.
Cornea and Sclera
The cornea and sclera were devoid of NPY immunoreactive fibers, also at the limbus and at the
intrascleral collector vessels. A few NPY immuno-
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Fig. 2. Adrenergic fibers in the
chamber angle of a normal albino
guinea pig. Note the high number
of fibers in the loose trabecular
tissue near the sclera (asterisk).
There is also a plexus of adrenergic
varicose fibers at the pigment epithelium (PE). The picture is included in order to facilitate comparison with the NPY immunoreactive fibers in Fig. 4 (X140).
reactive fibers could be seen around the episcleral
vessels at the limbus.
The Chamber Angle
The meshwork of the chamber angle is known to
contain many adrenergic fibers in guinea pigs,14 which
is in contrast with most other animals. The adrenergic
fibers are particularly common in the outermost part
of this tissue (Fig. 2). In contrast, it contained only
few, scattered NPY immunoreactive fibers, most in
parts well away from the sclera (Fig. 4).
The Iris
NPY immunoreactive fibers were
narrow zone immediately in front
muscle (Fig. 5). Only rarely were any
to penetrate into the muscle itself.
numerous in a
of the dilator
such fibers seen
They were less
common in the other parts of the iris stroma, decreasing in numbers anteriorly. NPY immunoreactive
fibers occurred in the adventitia of the major irideal
vessels (Fig. 6) but not in numbers comparable with
the adrenergic fibers.14 The NPY fibers did not seem
to be associated with capillaries or other small vessels.
Many of the immunoreactive stromal NPY fibers
appeared to be associated with melanophores in the
iris (Fig. 7), but some were without association with
any identifiable structure.
Nerve fiber trunks in the iris stroma occasionally
contained a small number of thin, varicose NPY
immunoreactive fibers (Fig. 6).
The sphincter was supplied with a small number
of essentially circumferentially running NPY immunoreactive fibers (Fig. 8). There was at times a
tendency for the NPY immunoreactive fibers to be
more numerous in the peripheral and posterior parts
of the muscle. The immunoreactive fibers did not
Fig. 3. Formaldehyde-induced
fluorescence
in
guinea-pig retina, 4 hr after
the intravitreal injection of
10 ^g alpha-methyldopamine. This treatment enhances the fluorescence of
the dopaminergic neurons,
which can be seen to form
two sublayers of fibers in
sublaminae 1 and 3 of the
inner plexiform, layer. There
are also four dopaminergic cell buuics i,arruwsj, one oi wnicn is sngnny oui or rocus. i ne picture is inciuaea to facilitate comparison with
the NPY immunoreactive fibers in Figures 11 and 12. Ph: photoreceptors; ONL: outer nuclear layer; OPL: outer plexiform layer; 1NL: inner
nuclear layer; IPL: inner plexiform layer (XI75).
Ph
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INVESTIGATIVE OPHTHALMOLOGY & VI5UAL SCIENCE / October 1984
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Fig. 4. NPY immunoreactive fibers of the trabecular region in the
chamber angle. There is a sparse
supply of NPY immunoreactive fibers (asterisk) in the middle of the
trabecular meshwork, and hardly
any next to the sclera. Compare
with Figure 2. IR: iris root; PE:
pigment epithelium; S: sclera. (Fluorescence micrograph X280).
seem to be associated with the occasional fine vessels
or melanophores in the muscle.
The Ciliary Body and the Ciliary Processes
NPY immunoreactive fibers were numerous in all
ciliary processes (Figs. 5, 9, 10). They ran close to
both blood vessels and the epithelium in the narrow
stroma of the ciliary processes. No intraepithelial
fibers were observed. No difference was found between
anterior and posterior ciliary processes. There was a
loose plexus of NPY immunoreactive fibers beneath
the epithelium of the ciliary body all the way back to
the ora serrata (Fig. 10).
The ciliary muscle contained a small-to-moderate
number of mainly circumferentially and some radially
directed delicate NPY immunoreactive fibers (Fig.
10). The number of such fibers did not appear dense
enough to ascertain that every muscle cell is reached
by a fiber. The number of fibers was roughly comparable with the number of adrenergic fibers.
The Choroid
The major arteries of the choroid were supplied
with a well-developed plexus of NPY immunoreactive
fibers, located at the border between the media and
the adventitia, precisely coinciding with the position
of the vascular adrenergic fibers (Fig. 11). However,
the immunoreactivity was less than that in nerves
around extraocular vessels. In addition, NPY immunoreactive fibers were common in the connective
Fig. 5. Low-power view of the NPY immunoreactive fibers of the dilator part of the iris and
the ciliary processes. Note that the NPY immunoreactive fibers occur in front of the dilator
muscle (D, arrow), in the iris stroma, and in the
ciliary processes (CP). (Fluorescence micrograph
X180).
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NPY NEURONS IN THE GUINEA-PIG EYE / Bruun er ol.
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Fig. 6. High-power micrograph
of NPY immunoreactive fibers in
the iris. Some occur in front of the
dilator muscle (D), a few around a
large vessel (V), and in the iris
stroma. A few also appear in a
nerve fibers bundle (NF). Above,
fluorescence micrograph; below,
phase contrast micrograph of the
same region (X450).
tissue in between the choroidal vessels, running without any apparent connection with any identifiable
structure. A few NPY immunoreactive fibers also
were observed in nerve fiber bundles. Preterminal,
nonvaricose immunoreactive fibers were not observed.
The Retina
Delicate, varicose NPY immunoreactive fibers were
seen in a narrow sublayer of intertwined fibers in
sublamina 1 of the inner plexiform layer, just at the
border to the inner nuclear layer (Fig. 11). Very
rarely, suchfibersalso could be seen in other sublayers
of the inner plexiform layer. Immunoreactive cell
bodies were seen in the innermost cell row of the
inner nuclear layer. They were quite difficult to detect
because their immunoreactivity often was concentrated to the hillock region with only very faint
immunofluorescence in the rest of the perikaryon
(Fig. 12). The nucleus was always devoid of immunofluorescence. The size of the cells was not estimated
readily because of their weak immunoreactivity but
was not distinctly different from the surrounding
amacrine cells. The frequency of the cells was for the
same reason difficult to estimate, but a judgement
based on the number of hillock-like aggregations of
immunoreactivity gave a figure of 5-10/mm section
length. The NPY immunoreactive cells occurred at
relatively regular intervals and were not seen in pairs
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Fig. 7. NPY immunoreactive fibers in
the iris stroma. Note that they tend to be
associated with melanophores. Above, fluorescence micrograph; below, phase contrast micrograph of the same region (X28O).
or clusters. They were not obviously different in
numbers in central or peripheral parts of the retina.
Cell processes could be seen to reach the sublayer of
NPY immunoreactive fibers in the inner plexiform
layer. Several hundred sections have been examined,
and an immunoreactive fiber was on two occasions
seen to penetrate the inner nuclear layer to reach the
outer plexiform layer. They were not seen to ramify
there, and no immunoreactive fibers were detected
in this layer. The NPY immunoreactive structures
described were not connected with any retinal blood
vessels.
Fig. 8. NPY immunoreactive fibers in the sphincter region
of the iris. Note that there are a few such fibers scattered
through the sphincter muscle (SP). Above, fluorescence micrograph; below, phase contrast micrograph of the same region
(XI80).
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Fig. 9. Equatorial section through
posterior ciliary processes. NPY immunoreactive fibers enter the ciliary
processes from the subepithelial network of the ciliary body. CB: ciliary
body; S: sclera. (Fluorescence micrograph X1I0).
The Optic Nerve
The optic nerve was not found to contain any
NPY immunoreactive fibers except an occasional
perivascular twig.
Sympathetic Denervation
No NPY immunoreactive fibers remained in the
ipsilateral anterior uvea 1 week after sympathetic
denervation. However, the choroid and the most
posterior quarter or fifth of the ciliary body showed
a decreased but still significant number of NPY
immunoreactive fibers after the operation (Fig. 13).
The fibers were seen in all locations described above
in the choroid, with a uniform decrease in their
number. The immunoreactivity of the fibers seemed
decreased.
No changes were seen in the NPY immunoreactive
fiber density on the contralateral control side or in
the retina on either side.
The Superior Cervical Ganglion
The superior cervical ganglion, was included in the
study in order to verify the origin of the NPY
immunoreactive fibers disappearing on sympathetic
denervation. It was seen to contain numerous cell
bodies displaying varying, weak-to-moderate fluorescence intensities. The size and shape of the NPY
immunoreactive neurons were not appreciably different from other neurons of the ganglion. There was
no apparent connection between cell size and immunoreactivity. The NPY immunoreactive cells were
scattered evenly throughout the ganglion. A few NPY
immunoreactive fibers also were seen.
Fig. 10. Meridional section through
the posterior part of the ciliary body.
There is a well-developed plexus of
NPY immunoreactive fibers under the
ciliary epithelium (CE) but only few in
the ciliary body proper. S: sclera. (Fluorescence micrograph XI80).
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Ph
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Fig. 11. NPY immunoreactive fibers in the retina. There
is a thin layer of processes in
sublamina I in the inner plexiform layer and scattered fibers
in the choroid. Above, fluorescence micrograph; below, phase
contrast micrograph of the same
region. Designation of layers like
in Figure 3 (X280).
ONL
= OPL
_JNL
IPL
Discussion
Based on structural relationships, many neuropeptides can be grouped into families. NPY belongs to
the family that also comprises the pancreatic polypeptides and peptide YY (PYY).2 Immunologic crossreactivity therefore can be expected with these peptides
and have, indeed, been revealed in gut endocrine
cells with the antiserum used in this study8 as well as
in studies on the brain. 3 However, absorption of the
antibody with these peptides did not diminish the
immunoreactivity in the nerve fibers in the guineapig eye, which makes it likely that the material
demonstrated is NPY rather than the pancreatic
polypeptides or PYY.
In the retina, NPY was found in a subclass of
Fig. 12. Higher magnification of the
retina with an NPY immunoreactive cell
body. Its vitread part is particularly prominent (arrow). Left, fluorescence micrograph; right, phase contrast micrograph of
the same region. Designation of layers like
in Fig. 3 (X450).
IPL
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NPY NEURONS IN THE GUINEA-PIG EYE / Druun er al.
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Fig. 13. NPY immunoreactive
fibers around some large vessels in
the choroid, 14 days after the excision of the homolateral cervical
sympathetic ganglia. A significant
number of immunoreactive fibers
remain around the vessels and in
the stroma, although the number is
somewhat decreased compared with
the normal. There is material with
nonspecific fluorescence in the lumen of three vessels (v). (Fluorescence micrograph X280).
amacrine cells. Several such subclasses are now known,
different for various neuropeptides and different in
various species (reviews1011). However, the concentration of the NPY immunoreactivity to the hillock
region of the cell body departs somewhat from what
usually has been seen previously with other peptides,
which distribute more or less evenly throughout the
cytoplasm. The hillock region is where the Golgi
complex is often found. Presumably then, NPY is
accumulated into granules in the Golgi apparatus and
then efficiently transported out into the processes,
whereas only little is transported to the perikaryon.
A similar accumulation of a regulatory peptide to the
Golgi region previously has been noted only in some
peripheral nerve cells.18
The NPY immunoreactive processes distribute almost exclusively to the outermost sublayer (sublamina
1) of the inner plexiform layer, which is the layer
where many of the dopaminergic fibers are found in
the guinea pig1519 (Fig. 3). It therefore is worth
noticing that NPY has been seen to coexist with a
catecholamine in the human medulla oblongata.20
However, dopaminergicfibersalso occur in sublamina
3 in the guinea-pig inner plexiform layer, which the
NPY immunoreactive fibers do not. It therefore seems
unlikely that NPY coexists with dopamine in the
retina. Other neurotransmitters known or presumed
in the guinea-pig retina have an even more different
pattern of distribution.''
The distribution of the NPY immunoreactive fibers
in the extraretinal parts of the eye is in many regions
identical with the distribution of the noradrenergic
sympathetic fibers originating in the superior cervical
ganglion14"1622 (see also Figs. 1, 2). The denervation
experiments prove that the NPY immunoreactive
fibers in the anterior uvea originate in the superior
cervical sympathetic ganglion, and, confirmingly, the
superior cervical ganglion was seen to contain numerous NPY immunoreactive cell bodies.
The similarity in fiber densities and distribution
makes it likely that NPY is present in most noradrenergic fibers of the guinea-pig uvea. Lundberg et
al6 obtained direct evidence for such a coexistence in
cats. They showed that the superior cervical ganglion
in cats contains many NPY immunoreactive neurons,
which also contain noradrenaline, as judged from
their content of tyrosine hydroxylase and dopamine
beta hydroxylase.
The observation that choroidal NPY immunoreactive fibers diminish in number but do not disappear
upon ablation of the cervical sympathetic ganglion
chain shows that in part they come from these
ganglia, but in part also from somewhere else. The
origin of the fibers remains to be determined.
The noradrenaline containing fibers in the guineapig uvea all originate in the superior cervical ganglion
chain,1415-2122 and the NPY immunoreactive fibers
that do not derive from this ganglion thus lack
noradrenaline. Conceivably therefore, NPY ocular
fibers can be divided into two classes, one where
it coexists with noradrenaline, and one where it
does not.
NPY immunoreactive fibers did not appear in
certain places where noradrenergic ones can be found,
most notably in the cornea and in parts of the
chamber angle. They were also fewer than the adren-
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / October 1984
ergic fibers at the iris dilator and around the major
vessels of the iris. Thus, also the noradrenergic fibers
can be subdivided into two classes, one with NPY
immunoreactivity and one without. Similarly, Lundberg et al6 have shown that the cells in the cat superior
cervical ganglion that contained NPY immunoreactivity were considerably fewer than the ones containing
tyrosine hydroxylase and dopamine beta hydroxylase,
ie, the NPY immunoreactive cells were fewer than
the adrenergic ones. They also noted that in several
tissues in the cat, there were significantly fewer NPY
immunoreactive fibers than adrenergic ones. The
observations reinforce the conclusion that only certain
noradrenergic neurons contain NPY.
NPY immunoreactive fibers occur around the arteries of the cat submandibular gland, and NPY has
been shown to give a long-lasting vasoconstriction in
this tissue, also after cervical sympathectomy. This
suggests that there are NPY-agonistic, vasoconstrictive
receptors, which are independent of sympathetic innervation.6 A different action also has been detected.
NPY is present in nerve fibers in the mouse vas
deferens. Here, it inhibited nerve-induced muscle
contractions but had no effect when the organ was
denervated sympathetically, suggesting that in this
case, it acts to inhibit the release of noradrenaline
from nerve terminals.4
The relative paucity of NPY immunoreactive fibers
around the iris blood vessels would suggest a minor
role on vessels in this region. However, the choroidal
vessels have a rich supply of NPY immunoreactive
fibers, and the vasoconstrictive effects therefore may
be more prominent in this part. It is worth noticing
that these fibers originate only in part in the cervical
sympathetic chain.
NPY has been associated previously with secretory
cells in, eg, the salivary glands,6 the pancreas, and
the gastrointestinal tract.8 It is, therefore, possible
that the fibers seen in the ciliary processes affect the
secretion of aqueous humour, and thus also the
intraocular pressure.
The density of NPY immunoreactive fibers in the
uvea is much higher than what has been found for
any other neuroactive peptide and is directly comparable with the density of noradrenergic fibers. It
therefore seems likely that NPY is present in most
noradrenergic neurons in the eye. Since the sympathetic adrenergic nervous system participates in the
regulation of the intraocular pressure and in the
inflammatory responses in rabbits,23"25 the as yet
unknown effects of NPY on these are of considerable
interest.
Key words: neuropeptide Y, guinea pig, uvea retina
Vol. 25
Acknowledgment
The APP was provided by Dr. J. R. Kimmel, Kansas
City, the BPP by Dr. R. E. Chance, Indianapolis, and the
NPY and PYY by Dr. K. Tatemoto, which is acknowledged
gratefully.
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